Human immunodeficiency virus (HIV) infection and related disease is a major public health problem worldwide. The retrovirus human immunodeficiency virus type 1 (HIV-1), a member of the primate lentivirus family (DeClercq E (1994) Annals of the New York Academy of Sciences, 724:438-456; Barre-Sinoussi F (1996) Lancet, 348:31-35), is generally accepted to be the causative agent of acquired immunodeficiency syndrome (AIDS) Tarrago et al FASEB Journal 1994, 8:497-503). AIDS is the result of repeated replication of HIV-1 and a decrease in immune capacity, most prominently a fall in the number of CD4+ lymphocytes. The mature virus has a single stranded RNA genome that encodes 15 proteins (Frankel et al (1998) Annual Review of Biochemistry, 67:1-25; Katz et al (1994) Annual Review of Biochemistry, 63:133-173), including three key enzymes: (i) protease (Prt) (von der Helm K (1996) Biological Chemistry, 377:765-774); (ii) reverse transcriptase (RT) (Hottiger et al (1996) Biological Chemistry Hoppe-Seyler, 377:97-120), an enzyme unique to retroviruses; and (iii) integrase (Asante et al (1999) Advances in Virus Research 52:351-369; Wlodawer A (1999) Advances in Virus Research 52:335-350; Esposito et al (1999) Advances in Virus Research 52:319-333). Protease is responsible for processing the viral precursor polyproteins, integrase is responsible for the integration of the double stranded DNA form of the viral genome into host DNA and RT is the key enzyme in the replication of the viral genome. In viral replication, RT acts as both an RNA- and a DNA-dependent DNA polymerase, to convert the single stranded RNA genome into double stranded DNA. Since virally encoded Reverse Transcriptase (RT) mediates specific reactions during the natural reproduction of the virus, inhibition of HIV RT is an important therapeutic target for treatment of HIV infection and related disease.
Until 1995, the only drugs approved in the United States were nucleoside inhibitors of RT (Smith et al (1994) Clinical Investigator, 17:226-243). Since then, two new classes of agents, protease inhibitors and non-nucleoside RT inhibitors (NNRTI), and more than ten new drugs have been approved (Johnson et al (2000) Advances in Internal Medicine, 45 (1-40; Porche D J (1999) Nursing Clinics of North America, 34:95-112). There are now three classes of drugs available: (1) the original nucleoside RT inhibitors, (2) protease inhibitors, and (3) the non-nucleoside RT inhibitors (NNRTI). Nucleoside RT inhibitors include zidovudine, didanosine (NIH), zalcitabine (NIH), lamivudine (BioChem Pharma Inc) and abacavir (Glaxo Wellcome plc). See Johnson V A (1995) Journal of Infectious Diseases, 171 :Suppl 2:S140-S149; Venrura et al (1999) Archives of Virology, 144:513-523; and Venrura et al Archives of Virology 1999, 144 (513-523). Approved protease inhibitor drugs include saquinavir (Hoffmann-La Roche Inc, Noble et al (1996) Drugs, 52:1, 93-112), ritonavir (Abbott Laboratories), indinavir (Merck & Co Inc), nelfinavir (Agouron Pharmaceuticals Inc) and amprenavir (Vertex Pharmaceuticals Inc). Approved NNRTI include nevirapine (Boehringer Ingelheim Corp, Grob et al (1992) AIDS Research and Human Retroviruses, 8:145-152; Pollard et al (1998) Clinical Therapeutics, 20:1071-1092), delavirdine (Pharmacia & Upjohn Inc, Freimuth W W (1996) Advances in Experimental Medicine and Biology, 394:279-289) and efavirenz (DuPont Pharmaceuticals Co, Adkins et al (1998) Drugs, 56:6, 1055-1066). Capravirine is an orally administered NNRTI therapeutic candidate (Brown W. (2000) Current Opinion in Anti-Infective Investigational Drugs 2(3):286-94).
RT can be inhibited by both nucleoside and non-nucleoside drugs (Venrura et al (1999) Archives of Virology, 144:513-523; Matthee et al (1999) Planta Medica 65:493-506). The nucleoside inhibitors act as competitive inhibitors, competing with the natural substrates or as chain terminators (Mayers D (1996) AIDS 10:Suppl 1, S9-S13; Villahermosa et al (1997) Biochemistry, 36:13223-13231; Klarmann et al (2000) Journal of Biological Chemistry, 275:359-366). The nucleoside inhibitors, including zidovudine, didanosine and zalcitabine, remain first-line therapies against HIV-1. However, extended use of these drugs leads to the development of HIV variants that are resistant to them (Moyle G J (1997) Journal of Antimicrobial Chemotherapy, 40:6, 765-777; Smith et al (1994) Clinical Investigator 17:226-243). This development of resistance has been associated with specific point mutations in the HIV pol gene, encoding RT.
The non-nucleoside inhibitors act by interacting with a non-substrate-binding site on the enzyme, i.e. allosterically (Proudfoot J R (1998) Current Opinion in Therapeutic Patents, 8:8, 971-982; DeClercq E (1998) Antiviral Research 38:3, 153-179; DeClercq E (1999) Farmaco 54:1-2, 26-45; Katlama C (1999) International Journal of Clinical Practice, 103:Suppl 16-20; Pederson et al (1999) Antiviral Chemistry and Chemotherapy 10:258-314). The NNRTI drugs have now gained a place in the arsenal of treatments for HIV-1 infection (Spence et al (1995) Science 267:988-993), acting non-competitively by interacting with a specific site on the RT that is near to, but distinct from, the active site where the nucleoside inhibitors bind. Several relevant crystal structures of HIV-1 RT complexed with the non-nucleoside inhibitors have been reported, expanding the understanding of how these inhibitors operate (Schafer-W et al (1993)Journal of Medicinal Chemistry 36:726-732).
Although drugs targeting reverse transcriptase and protease are in wide use and have shown effectiveness, particularly when employed in combination, toxicity and development of resistant strains have limited their usefulness (Palella, et al N. Engl. J. Med. (1998) 338:853-860; Richman, D. D. Nature (2001) 410:995-1001).
Combination therapy with RT inhibitors has proven to be highly effective in suppressing viral replication to unquantifiable levels for a sustained period of time. Also, combination therapy with RT and Prt inhibitors have shown synergistic effects in suppressing HIV replication. Unfortunately, 30 to 50% of patients currently fail combination therapy due to the development of drug resistance, non-compliance with complicated dosing regimens, pharmacokinetic interactions, toxicity, and lack of potency. Therefore, there is a need for new HIV-1 inhibitors that are synergistic in combination with other HIV inhibitors.
Assay methods capable of determining the presence, absence or amounts of HIV RT are of practical utility in the search for inhibitors as well as for diagnosing the presence of HIV.
Inhibition of HIV RT is an object of the invention. Inhibitors of HIV RT are useful to limit the establishment and progression of infection by HIV as well as in diagnostic assays for HIV RT, both of which are further objects of the invention. Preparation of compositions capable of inhibiting HIV RT is also an object of the invention.
There is a need for HIV RT inhibitors having improved antiviral and pharmacokinetic properties, including enhanced activity against development of HIV resistance, improved oral bioavailability, greater potency and extended effective half-life in vivo. New HIV RT inhibitors should be active against mutant HIV strains, have distinct resistance profiles, fewer side effects, less complicated dosing schedules, and orally active. In particular, there is a need for a less onerous dosage regimen, such as one pill, once per day.
Improving the delivery of drugs and other agents to target cells and tissues has been the focus of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the inhibitory drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g. to neighboring cells, is often difficult or inefficient.
Most agents currently administered to a patient parenterally are not targeted, resulting in systemic delivery of the agent to cells and tissues of the body where it is unnecessary, and often undesirable. This may result in adverse drug side effects, and often limits the dose of a drug (e.g., cytotoxic agents and other anti-cancer or anti-viral drugs) that can be administered. By comparison, although oral administration of drugs is generally recognized as a convenient and economical method of administration, oral administration can result in either (a) uptake of the drug through the cellular and tissue barriers, e.g. blood/brain, epithelial, cell membrane, resulting in undesirable systemic distribution, or (b) temporary residence of the drug within the gastrointestinal tract. Accordingly, a major goal has been to develop methods for specifically targeting agents to cells and tissues. Benefits of such treatment includes avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells.
Intracellular targeting may be achieved by methods and compositions which allow accumulation or retention of biologically active agents inside cells.